Immunoassay is an established biospecific assay method and is widely used in routine diagnostics and research laboratories. Another group of biospecific assays is DNA and RNA hybridization assays, although they are still under development. Two biospecific probes, a primary probe and a secondary probe (i.e., an antibody, DNA or RNA probe) are usually used in biospecific assays. They are both bound to the specific determinants of the analyte molecules and form a complex of three molecules (sandwich structure). Normally, one of these two reagents is labelled. Nowadays, the most commonly used labels are radioisotopes, enzymes, luminescent and fluorescent labels. Later in the text, the label used in a biospecific reaction will be referred to as a photoluminescent label, which denotes labels that generate or catalyse fluorescence, phosphorescence, chemiluminescence or bioluminescence.
There is a constantly increasing need for multiparameter analytics within routine diagnostics. Unfortunately, the existing techniques do not allow the use of more than two or three labels to be measured simultaneously because the spectrometric signals from different labels can not be sufficiently separated. The emission spectra of the photoluminescent labels overlap significantly and as a consequence they provide inadequate separation of different analytes over a required concentration range.
The purpose of this invention is to present a better method for multiparameter biospecific assays. The method according to this invention is based on methods that are generally known within the fields of immunology and DNA hybridization. Normally, they are performed as follows. The method uses two biospecific probes that recognize the analyte molecule k. In this text, these probes are referred to as the primary probe Ab(k,1) and the secondary probe Ab(k,2). When the secondary probe is labelled, for example, with a photoluminescent label F, it is denoted with the symbol Ab.sup.F (k,2). In the reaction solution, there is an excess of primary and secondary probes compared to the number of analyte molecules M.sub.k. When the analyte molecule, which is either a polypeptide or a macromolecule, has separate epitopes i.e. molecule structures that bind specifically to the probes, they form a complex Ab(k,1)+M.sub.k +Ab.sup.F (k,2). In principle, the amount of complex formed is directly proportional to the amount of the analyte, and the excess of primary and secondary probes remain in the solution. The complexes are separated from the free probes using a commonly known technique, for example, in which the primary probe is bound to a solid carrier and the free probes are washed away from the sample. Finally, the signal of bound label F in the complexes is measured in a traditional way which depends on the label chosen. The intensity of the signal obtained is directly proportional to the amount of label in the solution, and the response of the system is linear.
If the analyte to be measured is a small molecule without two or more epitopes which specifically bind to the probes, one can use a secondary probe that reacts specifically with the complex formed by the analyte and the primary probe (C. H. Self & al., Clin Chem 40 (1994) 2035-2041).
Principles of a multiparameter biospecific assay have been published earlier. It has been common practice to use multiple labels to tag biospecific reagents and to perform the separation of the signals on the basis of their different emission spectra. In most cases, however, the known multiparameter methods are based on the use of a solid support where the biospecific reagents can be immobilized at separate and optically distinguishable areas, or they are based on the use of artificial microparticles as a solid support. Some of the methods are reviewed below:
1. A method, in which various biospecific probes are attached to a matrix, which is formed by small areas on a planar solid support, is described in the patent PCT WO 84/01031. In this method, after the reaction and the wash, the signals from the photoluminescent labels in each area are measured separately, for example, using a laser scanning microscope.
2. A method, in which the identification of the analyte category is based on the color of the microparticles, which are used as a solid support and which is achieved by optically measuring the light absorption of the particle to be analyzed (J. G. Streefkerk & al., Protides Biol. Fluids 24 (1976) 811-814 and U.S. Pat. No. 5,162,863).
3. A method, in which the identification of the analyte category is performed by optically measuring the absorption of the dye inside the particle, the refractive index or the size of the particle to be analyzed (U.S. Pat. No. 5,162,863).
4. A method, in which the identification of the analyte category is based on the use of different particle sizes and in which the identification is performed by optically measuring the diameter of the particle to be analyzed (T. M. McHugh & al., Journal of Immunological Methods 95 (1986) 57-61).
5. A method, in which the microparticles are identified by means of fluorescent dyes that are mixed or impregnated within the particles, and the biospecific signal is measured from the fluorescence intensity of another fluorescent dye, such as FITC (EP 126450, GOIN 33/58).
6. A method, in which a dye emitting short decay time fluorescence (decay time a few nanoseconds) is used for the identification of microparticles, and a dye emitting long decay time fluorescence (decay time from 10 microseconds to 2 milliseconds) is used for measuring the analyte concentrations, and in which a time resolved fluorometer is used for the discrimination of the short and long life time fluorescence (U.S. Pat. No. 5,028,545).
7. A method, in which a dye emitting short decay time fluorescence (decay time a few nanoseconds) is used for the identification of the microparticles, and a molecule which generates chemiluminescence or bioluminescence (decay time several seconds) is used to measure the analyte concentrations, and in which the fluorescence and luminescent signal can effectively be separated from the fluorescence because they are excited and they emit light at different times (FI-patent 89837).
8. A method, in which a dye emitting short decay time fluorescence (decay time a few nanoseconds) is used for the identification of the microparticles and a dye emitting phosphorescence, (decay time from 10 microseconds to 2 milliseconds) is used to measure the analyte concentrations, and in which a time resolved fluorometer is used for the discrimination between the short decay time fluorescence and the long decay time phosphorescence (FI-patent 90695).
9. A method, in which dyes emitting long decay time fluorescence, such as fluorescent chelates of lanthanide ions Tb, Dy, Eu and Sm, are used for the identification of the microparticles and for measurement of the biospecific signal (FI-application 931198).
A common problem in many multiparameter assays mentioned above is that the signal of the photoluminescent label, which indicates the analyte category, and the signal from the photoluminescent label, which measures the concentration of the biospecific probe, interfere with each other. This is a problem that significantly restricts the dynamic range of the measurement of the analyte concentration. It is essential for the sensitivity of the method of this invention, as well as for the sensitivity of the multiparameter assays mentioned above and previously known, that the signal from the indicator used for the identification of the analyte does not interfere with the signal from the photoluminescent label used for the measurement of the biospecific reaction. This interference may become particularly significant when measuring low analyte concentrations and when a wide dynamic range is required for the measurement of the biospecific signal. In methods 6, 7, and 8 referred to above, interference is eliminated by choosing such photoluminescent labels for the measurement of the biospecific reaction and identification labels which have substantially different emission decay times. In methods 1, 2, 3 and 4 the analyte is identified using an alternative method rather than using a photoluminescent label. In methods 5 and 9, the identification method of the analyte essentially restricts the dynamic range of the measurement.
Another problem with methods 6, 7, 8 and 9 mentioned above is the long measurement time, caused by the long decay time (T1/2=1 millisecond) of the fluorescent and phosphorescent labels. This is due to the saturation of the excited states of the labels, which restricts the intensity of the exciting light to such a low level that a measurement time of up to one second is needed for each microparticle.
Likewise, the measurement of the signals from labels that are based on chemiluminescence, bioluminescence and electroluminescence, also take at least one second.